Diagnostic device



United States Patent 3,212,855 DIAGNOSTIC DEVICE Ray Mast, John Rebar,Jr., and Joseph Fraser, Elkhart, Ind., assignors to Miles Laboratories,Inc., Elkhart, Ind., a corporation of Indiana N0 Drawing. Filed Aug. 6,1962, Ser. No. 214,867 7 Claims. (Cl. 23253) This invention relates toan improved diagnostic composition and to a method for its preparation.In particular, this invention is concerned with a diagnostic test usefulin qualitative detection and quantitative determination of ketone bodiesin body fluids, especially acetoacetic acid (beta-ketobutyric acid) inurine. More particularly, this invention is concerned with a diagnostictest reagent composition which is incorporated upon a bibulous carrier.

The fats utilized by the body normally undergo complete oxidation withformation of carbon dioxide and water. Since the fatty acid moleculeshave long chains of carbon atoms, it is theorized that a large number ofintermediate products are formed during the oxidation and under normalcircumstances these are rapidly further oxidized. Findings in the fieldof fat metabolism suggest that during the course of oxidation of thefatty acid chain the carbon atoms are split off in pairs, or in otherwords, oxidation takes place at the ,B-carbon atom. In certain abnormalphysical conditions the oxidation of fat is incomplete and certainproducts such as acetoacetic acid appear in the urine. These substancesare called ketone bodies and their appearance creates a condition whichis called ketosis. Ketosis occurs typically in diabetes mellitus, but italso occurs as a result of other abnormal conditions, e.g., fasting,hyperpituitary activity, etc. Under these abnormal conditions ketonebodies tend to accumulate in the blood and, because the rental thresholdfor them is low, they appear in the urine. The healing arts have longrecognized the usefulness of tests for ketone bodies in the urine, henceit is considered extremely desirable to provide a simple and economicaltest for the qualitative and quantitative determination of ketone bodiesin the urine which may be advantageously used by the laboratorytechnician as well as the physician.

A variety of reagents and techniques have been used or proposed in thepast for the detection of ketone bodies in urine. A number of suchreagents and techniques have involved the use of a water solublenitroprusside as a reactive ingredient or agent. In one particularreagent formulation, the nitroprusside reaction is carried out in thepresent of ammonia in order to develop particular colorations (see, forexample, U.S. Patent 2,186,902 to Fortune). An improvement over theFortune-type formulation is disclosed in US. Patent 2,509,140 to AlfredH. Free, and assigned to the assignee of the present application' Thispatent discloses formulations for the detection of ketone bodies inurine which contain water soluble nitroprusside, an aliphatic amino acidand an alkaline material. It was found, according to the patent, thatwhen a soluble nitroprusside is present in alkaline solution with analiphatic amino acid, e.g., glycine, a diagnostic composition isprovided which is particularly adapted for the detection of ketonebodies in urine without evolution of ammonia.

An improvement of the foregoing test composition is described andclaimed in U.S. Patent 2,577,978, issued December 11, 1951, to Nichollsand Fonner and assigned to the assignee of the present application. Itwas discovered by these patentees that incorporation of lactose orsimilar sugars into the diagnostic composition of US. Patent 2,509,140greatly enchanced the utility and reliability of the diagnosticcomposition.

3,212,855 Patented Oct. 19, 1965 'ice A still further improved testcomposition is described in US. Patent 2,990,253, issued June 27, 1961,to Robert R. Smeby and assigned to the assignee of the presentapplication, which provides a test composition in the form of bibulousstrips or sticks. However, because of the instability of nitroprussidein an aqueous alkaline medium, the nitroprusside must be kept separatednot only during the impregnation of the carrier but until such time asthe test is ready for use. A method was discovered by the patentee ofUS. 2,990,253 of achieving the necessary separation, which separationwas etfected by first ap plying the nitroprusside to the carrier in anacidic, aqueous medium thus preserving the stability of the compoundand, after drying, dipping the carrier into a nonaqueous solution oforganic bases such as various amines or aminoalcohols to achieve thenecessary alkalinity.

The volatility and hygroscopicity of the amine constituents of the priorart formulations, however, are undesirable features of that test.Further, the selection of amines or aminoalcohols or mixtures thereof isrendered difiicult in that all amines and aminoalcohols are notoperable. In addition, in all of the prior compositions and methods, ithas been difiicult if not substantially impossible to protect thenitroprusside ingredients from the deleterious effects of moisture andalkaline sodium phosphates during storage.

\Vhile the foregoing discussed patents have contributed greatly to theadvancement of the art of diagnosing for ketonuria and otherdisturbances of metabolism evidenced by the presence of ketone bodies inthe urine and the advances made have been worthwhile, none havecompletely solved the problem of the instability of sodium nitroprussidein an aqueous alkaline medium. Nitroprusside is stable only at a pHbelow 7 and is operable only in an alkaline medium at a pH over 8. Inother words, most of the nitroprusside is destroyed so that noperceptible reaction with acetoacetic acid can be obtained under thosecircumstances. The commercial diagnostic methods made available inaccordance with the disclosures thereof have, however, aided thephysicians and clinicians in the diagnoses and control of the causes ofketonuria.

To summarize, the prior art teaches the use of a water solublenitroprusside, an aliphatic amino acid and an alkaline phosphate bufferas essential ingredients of a test for ketone bodies and teaches thepreparation of such diagnostic compositions in the form of bibulousstrips or sticks and a method of preparing same.

From a commercial point of view the test compositions in the form ofbibulous strips or sticks are highly preferred for the reason that suchprovide the diagnostician with a simple dip and read test. Such simpledip and read tests provide many advantages over prior known liquid ortabletted reagent compositions from the standpoint of absence ofcumbersome equipment, ease and simplicity of test procedure, ease ofdisposal of test devices and rapidity of test procedure, to mention afew of the advantages.

In accordance with this invention, we have discovered an improveddiagnostic composition and method of preparing such ketone diagnosticcomposition in strip or stick form which successfully overcomes thehereinabove enumerated disadvantages of the prior known compositions.

More specifically, we have discovered an improved composition for aketone diagnostic in stick form which is tremendously stable, andresistant to the deteriorative effects of moisture and the alkalinesodium phosphates. In addition, we have discovered an improved methodfor preparing an improved diagnostic in strip or stick form comprising atwo-step procedure which involves initially treating the bibulouscarrier with 3, an aqueous phosphate-containing formulation and,secondly, impregnating the thusly treated carrier with a novelformulation comprising sodium nitroprusside and an organic film-formingpolymer.

Among the numerous advantages provided by this invention, one is thatthe first step of the preparation may be carried out well ahead of thesecond step, i.e., the carrier impregnated with the phosphate-containingcomposition may be prepared and stored for periods of time prior toimpregnation with the second formulation.

Broadly, the initial treating formulation comprises a bufier, providinga pH range of about 8 10, and an amino acid. By way of example ofbuffering systems useful in the compositions of this invention aretriand disodium phosphates, borates, citrates, carbonates, ethylenediamine tetraacetate (sodium salt), etc. While any water soluble aminoacid may be used in the compositions of this invention, in the preferredembodiment the amino acid is selected from the group of glycine andalanine.

The second treatment formulation comprises alkali metal nitroprusside,an organic film-forming compound and an organic solvent. The organicfilm-forming compounds called for in the compositions of this inventionmay be any organic film-forming compound which is soluble in thecommonly used organic solvents, does not exhibit strong bufferingcapacity, and has a pH on the acid side, for example,polyvinylpyrrolidone-vinyl acetate copolymers; vinyl pyrrolidone-styrenecopolymers; water solutions of acrylic copolymers; coploymers of methylvinyl ether and maleic anhydride; polyethylene glycol; polyvinylacetate; and interpolymers of methyl vinyl ether and maleic anhydride.From an economic standpoint and ease of handling, however, copolymers ofpolyvinylpyrrolidone-vinyl acetate are preferred. It is readily seenthat the selection of an organic filmforming compound meeting therequirements of this invention is dictated solely by economicconsiderations.

Among the organic solvents found suitable for use in the compositionsand method of this invention are dimethyl sulfoxide, methanol, ethanoland dimethyl formamide and mixtures thereof. In addition to theforegoing ingredients, we have found that it is desirable but notessential to include such diluent substances as chloroform, carbontetrachloride, benzene, etc. and a wetting agent, for example, aerosol,diglycol laurate and organic phosphate esters of anionic detergents inethanol which are known commercially as Gafac RE610 and Gafac RESIO, andmixtures thereof. The diluent substances are useful to reducehygroscopicity of the testing reagents, while the wetting agent aids inproducing an even diffusion of color on the diagnostic stick.

The following examples will illustrate the improved diagnosticcomposition of the present invention, the scope of the invention not,however, being limited to the specific details of these examples:

Example ].Frmulati0n of the impregnating solutions Na PO .12H O g 210Disodium phosphate, anhydrous g 90 Glycine g 187 Distilled water to ml1000 Sodium nitroprusside, anhydrous g 8 Polyvinyl pyrrolidone/ vinylacetate copolymer (50% in ethanol) ml 65 Dimethyl sulfoxide ml 380Anhydrous ethanol ml 185 Chloroform ml 350 Organic phosphate ester ofanionic detergent ml 17 PREPARATION OF IMPREGNATING SOLUTIONS SolutionA.-210 grams of trisodium phosphate, 90

grams of disodium phosphate and 187 grams of glycine were mixed togetherin the dry state. 750 ml. of boiling hot distilled water were added tothe dry mixture and stirred until solution occurred.

Solution B.8 grams of sodium nitroprusside were measured into a oneliter volumetric flask. To this was added 65 ml. ofpolyvinylpyrrolidone/vinyl acetate copolymer and 185 ml. of anhydrousethanol and the solution mixed thoroughly. 380 ml. of dimethyl sulfoxidewere then added to the mixture with stirring until the nitroprusside wassolubilized. 350 ml. of chloroform, 17 ml. of a 10% anionic detergent(organic phosphate ester) in anhydrous ethanol were then added to thesolution.

PREPARATION OF REAGENT STRIPS Bibulous sticks, that is, absorbent paperout into narrow strips having dimensions of about 3" x /s" x 0.029,imprinted with a water impervious barrier portion of about from the tipwere dipped into impregnating Solution A, followed by drying in a dryingtunnel at a temperature of about 100 C., for 13 minutes. After drying,the strips were similarly dipped into Solution B and dried at about C.for 11 minutes in a forced draft oven. The finished impregnated stripsare light buff in color.

In preparing the formulations for use in the diagnostic strips of thisinvention, We have found the optimum ranges of essential ingredients tobe about 0.525 grams sodium nitroprusside; ll.7585.0 grams amino acid;l8.8940.0 grams buffer, comprising trisodium phosphate in the range ofabout 13.2657.0 grams and a range of about 5.6282.5 grams disodiumphosphate; and 4.1202.5 grams organic film-forming material.

The following examples are illustrative of other formulations preparedin accordance with this invention:

Example 2 Solution A:

Na HPO anhydrous gm 58.4 Glycine gm 20.0 Distilled water ml 180.0Solution B:

Sodium nitroprusside gm 1.0 Anhydrous methanol ml 100.0 Diglycol laurateml 1.0 Polyvinylpyrrolidone/vinyl acetate copolymer (50% in ethanol) ml20.0

Example 3 Solution A:

Sodium borate gm 5.0 Glycine gm 10.0 Distilled water ml 100.0 SolutionB:

Sodium nitroprusside gm 0.5 Anhydrous methanol ml 9.0 Anhydrous ethanolml 40.0 Polyvinylpyrrolidone/ vinyl acetate copolymer (50% in ethanol)ml 3.0 Diglycol laurate ml 2.0

Example 4 Solution A:

Glycine gm 25.0 Na CO gm 30.0 Distilled water ml 100.0 Solution B:

Sodium nitroprusside gm 0.5 Anhydrous methanol ml 9.0 Anhydrous ethanolml 40.0 Polyvinylpyrrolidone/vinyl acetate copolymer (50% in ethanol) ml3.0 Diglycol laurate ml 2.0

Example 5 Solution A:

Glycine gm E t h y l e n e diamine tetraacetate (sodium salt) "gm"Distilled water ml Solution B:

Sodium nitroprusside gm Polyvinylpyrrolidone/vinyl acetate copolyrner(50% in ethanol) ml Anhydrous ethanol ml Dimethylsulfoxide ml Chloroformml Aerosol (25 in ethanol ml Organic phosphate ester of anionicdetergent in ethanol ml Example 6 Solution A:

Glycine gm N33P04.12H2O gl'I1 Na HPO anhydrous gm Distilled water mlSolution B:

Sodium nitroprusside gm Anhydrous ethanol ml Dimethylsulfoxide mlChloroform ml Polyvinyl acetate ml Organic phosphate ester of anionicdetergent (10%) in ethanol ml Aerosol (25%) in ethanol ml Example 7Solution A:

Glycine "gm-.. Na3PO4.12H2O gm Na HPO anhydrous gm Distilled Water mlSolution B:

Sodium nitroprusside gm Anhydrous ethanol ml Dimethylsulfoxide mlChloroform ml Organic phosphate ester of anionic detergent (10%) inethanol ml Aerosol (25%) in ethanol ml Interpolymer of methyl vinylether and maleic anhydride gm Example 8 Solution A:

Glycine gm N33PO4-12H2O grn Nag-IP0 anhydrous gm Distilled water mlSolution 13:

Sodium nitroprusside gm Anhydrous methanol ml Aerosol (25%) in ethanolml Ethyl cellulose gm Anhydrous ethanol ml Example 9 Solution A:

Glycine --gm Na PO .12I-I O gm NilgHPO gm Distilled water ml Solution B:

Sodium nitroprusside gm Polyvinylpyrrolidone/vinyl acetate copolymer mlAnhydrous ethanol gm Ethyl lactate ml Chloroform ml Organic phosphateester of anionic detergent (10%) in ethanol ml Aerosol (25%) in ethanolml The preparation of the impregnating solutions and reagent stripsbased on the foregoing Examples 2 through 9 are carried out in themanner described in Example 1.

In use, an impregnated strip prepared as described above is dipped inthe liquid specimen to be tested. When contacted with a fluid specimencontaining ketone bodies, the test strip will give a positive colorreaction. The color resulting on the strip is then compared with aprecalibrated color chart for determination of the quantitative amountof ketone bodies contained on the specimen tested. The color developedon the strips in the presence of ketone bodies varies in intensityaccording to the amount of ketone bodies present in the specimen, i.e.,from very light purple indicating the presence of 10-20 mg. percent ofketonebodies to a very dark purple indicating over mg. percent.Utilizing the diagnostic strips of this invention, a positive colorreaction will de velop within 15 to 30 seconds in the presence of ketonebodies.

It is to be understood that other bibulous materials, e.g., small sticksof wood, etc., as well as other methods for applying the impregnatingsolutions to the test strips and for drying the thus impregnated stripsmay also be employed.

It is obvious that certain changes may be made in the above compositionsand methods without departing from the spirit and scope of the inventionand it is intended that all matter contained in the foregoingdescription shall be interpreted as illustrative and not in a limitingsense. It is also understood that other modifications may be madeWithout departing from the spirit and scope of the appended claims.

We claim:

1. A process for the preparation of a test device for the detection ofketone bodies in body fluids which comprises:

(A) impregnating a bibulous carrier with an aqueous solution of a bufferproviding a pH range of from about 8 to about 10 and a water solubleamino acid,

(B) drying the impregnated bibulous carrier;

(C) further impregnating the bibulous carrier in the area previouslyimpregnated with the buffer and amino acid with a solution, in anorganic solvent, of

(1) an alkali metal nitroprusside, and

(2) a polymeric substance selected from the group consisting ofpolyvinylpyrrolidone-vinyl acetate copolymers, methyl vinyl ether-maleicanhydride copolymers, polyethylene glycol, polyvinyl acetate, methylvinyl ether-maleic anhydride interpolymers, vinyl pyrrolidone-styrenecopolymers and water soluble acrylic copolymers; and

(D) removing the solvent from the further impregnated bibulous carrier.

2. A process as in claim 1 wherein the amino acid is selected from thegroup consisting of glycine and alanine.

3. A process as in claim 1 wherein the buffer is a mixture of disodiumphosphate and trisodium phosphate.

4. A process as in claim 1 wherein the solvent is selected from thegroup consisting of dimethyl sulfoxide, methanol, ethanol, dimethylformamide and mixtures thereof.

5. A process as in claim 1 wherein the organic filmforming polymericsubstance has a pH on the acid side.

6. A test device for the detection of ketone bodies in body fluidsprepared by a process which comprises:

(A) impregnating a bibulous carrier with an aqueous solution of a butterproviding a pH range of from about 8 to about 10 and a water solubleamino acid;

(B) drying the impregnated bibulous carrier;

(C) further impregnating the bibulous carrier in the area previouslyimpregnated With the butter and amino acid with a solution, in anorganic solvent, of

(1) an alkali metal nitroprusside, and

7 8 (2) a polymeric substance selected from the References Cited by theExaminer group consisting of polyvinylpyrrolidone-vinyl UNITED STATESPATENTS acetate copolymers, methyl vinyl ether-maleic anhydridecopolymers, polyethylene glycol, poly- 2186902 1/40 Fortunevinylacetate, methyl vinyl ether-maleic anhy- 5 2229155 1/41 Wenker 23253dride interpolymers, vinyl pyrrolidone-styrene 250914O 5/50 l 23 230copolymers and water soluble acrylic copoly- 2577978 12/51 Nlchons et 23230 mars; and, 2,990,253 6/61 Smeby 23-253 (D) removing the solvent fromthe further impreg- 3092465 6/63 Adams et 23253 nated bibulous carrier.10 FOREIGN PATENTS 7. A test device as in claim 6 wherein the amino acid867192 5/61 Great Britain is selected from the group consisting ofglycine and alanine. MORRIS O. WOLK, Primary Examiner.

1. A PROCESS FOR THE PREPARATION OF A TEST DEVICE FOR THE DETECTION OFKETONE BODIES IN BODY FLUIDS WHICH COM PRISES: (A) IMPREGNATING ABIBULOUS CARRIER WITH AN AQUEOUS SOLUTION OF A BUFFER PROVIDING A PHRANGE OF FROM ABOUT 8 TO ABOUT 10 AND A WATER SOLUBLE AMINO ACID, (B)DRYING THE IMPREGNATED BIBULOUS CARRIER; (C) FURTHER IMPREGNATING THEBIBULOUS CARRIER IN THE AREA PREVIOUSLY IMPREGNATED WITH THE BUFFER ANDAMINO ACID WITH A SOLUTION, IN AN ORGANIC SOLVENT, OF (1) AN ALKALIMETAL NITROPRUSSIDE, AND (2) A POLYMERIC SUBSTANCE SELECTED FROM THEGROUP CONSISTING OF POLYVINYLPYRROLIDONE-VINYL ACETATE COPOLYMERS,METHYL VINYL ETHER-MALEIC ANHYDRIDE COPOLYMERS, POLYETHYLENE GLYCOL,POLYVINYL ACETATE, METHYL VINYL ETHER-MALEIC ANHYDRIDE INTERPOLYMERS,VINYL PYRROLIDONE-STYRENE COPOLYMERS AND WATER SOLUBLE ACRYLICCOPOLYMERS; AND (D) REMOVING THE SOLVENT FROM THE FURTHER IMPREGNATEDBIBULOUS CARRIER.